Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.
Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.
复制标题
MDR1-双顺反子逆转录病毒载体 Ha-MDR-IRES-gp91 体内 P-糖蛋白和 gp91 的药物选择共表达。
DOI：
10.1002/jgm.362
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Tsuruo,Takashi
中科院分区：
文献类型：
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作者：
Sugimoto,Yoshikazu;Tsukahara,Satomi;Sato,Shigeo;Suzuki,Mutsumi;Nunoi,Hiroyuki;Malech,HarryL;Gottesman,MichaelM;Tsuruo,Takashi
背景逆转录病毒转导人造血干细胞是基因治疗的一个有吸引力的策略，然而，转导效率和转基因表达的持续时间可能不令人满意，在目前的方案。人多药耐药基因（MDR 1）与治疗基因的共表达为转基因细胞提供了可选择的生长优势。方法构建了一种双顺反子逆转录病毒载体Ha-MDR-IRES-gp 91，用于MDR 1与X连锁慢性肉芽肿病（X-CGD）相关基因gp 91的共表达。药物选择性的P-糖蛋白和gp 91的共表达进行了评估transducedcells.ResultsEpstein-巴尔病毒转化B细胞与Ha-MDR-IRES-gp 91转导的X-CGD患者共表达人P-糖蛋白和gp 91，并获得超氧化物生成活性。用Ha-MDR-IRES-gp 91转导来自X-CGD患者的人CD 34阳性细胞，随后用2 ng/ml长春新碱处理。13天后，通过FACS分析，20%的Ha-MDR-IRES-gp 91-转导细胞为P-糖蛋白-和gp 91-阳性。转导群体的超氧化物生成活性是正常细胞的27%。移植Ha-MDR-IRES-gp 91-转导骨髓细胞的小鼠显示外周血单核细胞中P-糖蛋白和gp 91共表达。通过给予紫杉醇，所有4只受检小鼠中P-糖蛋白和gp 91阳性细胞的比例均增加。结论MDR 1-双顺反子载体可用于体内筛选转导的造血细胞。这可能导致干细胞基因治疗患者血细胞中转基因的持续表达。版权所有© 2003年约翰威利父子有限公司。
BackgroundRetroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols. Co‐expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells.MethodsA bicistronic retrovirus vector, Ha‐MDR‐IRES‐gp91, was constructed for the co‐expression ofMDR1 and gp91, a gene responsible for X‐linked chronic granulomatous disease (X‐CGD). Drug‐selected co‐expression of P‐glycoprotein and gp91 was evaluated in transduced cells.ResultsEpstein‐Barr virus‐transformed B cells from X‐CGD patients transduced with Ha‐MDR‐IRES‐gp91 co‐expressed human P‐glycoprotein and gp91, and acquired superoxide‐generating activity. Human CD34‐positive cells from an X‐CGD patient were transduced with Ha‐MDR‐IRES‐gp91 and subsequently treated with 2 ng/ml vincristine. After 13 days, 20% of Ha‐MDR‐IRES‐gp91‐transduced cells were P‐glycoprotein‐ and gp91‐positive by FACS analysis. The superoxide‐generating activity of the transduced population was 27% of that of normal cells. Mice transplanted with Ha‐MDR‐IRES‐gp91‐transduced bone marrow cells showed co‐expression of P‐glycoprotein and gp91 in peripheral blood mononuclear cells. By administering paclitaxel, the proportions of P‐glycoprotein‐ and gp91‐positive cells were increased in all the four mice examined. When mice transplanted with Ha‐MDR‐IRES‐gp91‐transduced cells were repeatedly administered paclitaxel, the ratios of P‐glycoprotein‐ and gp91‐positive cells were maintained for over 1 year.ConclusionsThese results suggest thatMDR1‐bicistronic vectors may be useful to select the transduced hematopoietic cellsin vivo. This may lead to the sustained expression of transgenes in the blood cells of patients treated with stem cell gene therapy. Copyright © 2003 John Wiley & Sons, Ltd.